FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2100904923> ?p ?o ?g. }

• W2100904923 endingPage "1019" @default.
• W2100904923 startingPage "1010" @default.
• W2100904923 abstract "Niemann-Pick type C1 disease (NPC1) is an inherited neurovisceral lipid storage disorder, hallmarked by the intracellular accumulation of unesterified cholesterol and glycolipids in endocytic organelles. Cells acquire cholesterol through exogenous uptake and endogenous biosynthesis. NPC1 participation in the trafficking of LDL-derived cholesterol has been well studied; however, its role in the trafficking of endogenously synthesized cholesterol (endoCHOL) has received much less attention. Previously, using mutant Chinese hamster ovary cells, we showed that endoCHOL moves from the endoplasmic reticulum (ER) to the plasma membrane (PM) independent of NPC1. After arriving at the PM, it moves between the PM and internal compartments. The movement of endoCHOL from internal membranes back to the PM and the ER for esterification was shown to be defective in NPC1 cells. To test the generality of these findings, we have examined the trafficking of endoCHOL in four different physiologically relevant cell types isolated from wild-type, heterozygous, and homozygous BALB/c NPC1NIH mice. The results show that all NPC1 homozygous cell types (embryonic fibroblasts, peritoneal macrophages, hepatocytes, and cerebellar glial cells) exhibit partial trafficking defects, with macrophages and glial cells most prominently affected.Our findings suggest that endoCHOL may contribute significantly to the overall cholesterol accumulation observed in selective tissues affected by Niemann-Pick type C disease. Niemann-Pick type C1 disease (NPC1) is an inherited neurovisceral lipid storage disorder, hallmarked by the intracellular accumulation of unesterified cholesterol and glycolipids in endocytic organelles. Cells acquire cholesterol through exogenous uptake and endogenous biosynthesis. NPC1 participation in the trafficking of LDL-derived cholesterol has been well studied; however, its role in the trafficking of endogenously synthesized cholesterol (endoCHOL) has received much less attention. Previously, using mutant Chinese hamster ovary cells, we showed that endoCHOL moves from the endoplasmic reticulum (ER) to the plasma membrane (PM) independent of NPC1. After arriving at the PM, it moves between the PM and internal compartments. The movement of endoCHOL from internal membranes back to the PM and the ER for esterification was shown to be defective in NPC1 cells. To test the generality of these findings, we have examined the trafficking of endoCHOL in four different physiologically relevant cell types isolated from wild-type, heterozygous, and homozygous BALB/c NPC1NIH mice. The results show that all NPC1 homozygous cell types (embryonic fibroblasts, peritoneal macrophages, hepatocytes, and cerebellar glial cells) exhibit partial trafficking defects, with macrophages and glial cells most prominently affected. Our findings suggest that endoCHOL may contribute significantly to the overall cholesterol accumulation observed in selective tissues affected by Niemann-Pick type C disease. Niemann-Pick type C (NPC) disease is a fatal autosomal recessive neurovisceral disorder characterized clinically by progressive neurodegeneration in the central nervous system (CNS) and hepatosplenomegaly. Biochemically, it is characterized by the intracellular accumulation of unesterified cholesterol and glycolipids within the endosomal/lysosomal system in cells of the liver, spleen, brain, etc. (1Pentchev P.G. Comly M.E. Kruth H.S. Vanier M.T. Wenger D.A. Patel S. Brady R.O. A defect in cholesterol esterification in Niemann–Pick disease (type C) patients.Proc. Natl. Acad. Sci. USA. 1985; 82: 8247-8251Google Scholar, 2Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E.D. Neufeld E.B. Blanchette-Mackie E.J. Pentchev P.G. Niemann–Pick disease type C: a lipid trafficking disorder.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th edition. Vol. 3. McGraw-Hill, New York2001: 3611-3633Google Scholar). The disease can be caused by mutations in one of two genetically distinct loci, NPC1 and NPC2 (3Steinberg S.J. Ward C.P. Fensom A.H. Complementation studies in Niemann–Pick disease type C indicate the existence of a second group.J. Med. Genet. 1994; 31: 317-320Google Scholar, 4Vanier M.T. Duthel S. Rodriguez-Lafrasse C. Pentchev P. Carstea E.D. Genetic heterogeneity in Niemann–Pick C disease: a study using somatic cell hybridization and linkage analysis.Am. J. Hum. Genet. 1996; 58: 118-125Google Scholar). The Npc1 gene encodes a 1,278 amino acid multipass transmembrane protein that contains a putative sterol-sensing domain (5Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene.Science. 1997; 277: 232-235Google Scholar, 6Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgens M.E. Comly M. Cooney A. Brown A. Kaneski C.R. Blanchette-Mackie E.J. Dwyer N.K. Neufeld E.B. Chang T.Y. Liscum L. Strauss III, J.F. Ohno K. Zeigler M. Carmi R. Sokol J. Marckis D. O’Neill R.R. van Diggelen O.P. Elleder M. Patterson M.C. Brady R.O. Vanier M.T. Pentchev P.G. Tagle P.A. Niemann–Pick C1 disease gene: homology to mediators of cholesterol homeostasis.Science. 1997; 277: 228-231Google Scholar). The Npc2 gene encodes a soluble protein known as HE1, a lysosomal protein that binds cholesterol with high affinity and that can be secreted into the growth medium (7Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Identification of HE1 as the second gene of Niemann–Pick C disease.Science. 2000; 290: 2298-2301Google Scholar, 8Kirchhoff C. Osterhoff C. Young L. Molecular cloning and characterization of HE1, a major secretory protein of the human epididymis.Biol. Reprod. 1996; 54: 847-856Google Scholar). Mutations in Npc1 account for 95% of all cases of NPC disease, whereas mutations at NPC2 account for the remaining 5% (5Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene.Science. 1997; 277: 232-235Google Scholar, 6Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgens M.E. Comly M. Cooney A. Brown A. Kaneski C.R. Blanchette-Mackie E.J. Dwyer N.K. Neufeld E.B. Chang T.Y. Liscum L. Strauss III, J.F. Ohno K. Zeigler M. Carmi R. Sokol J. Marckis D. O’Neill R.R. van Diggelen O.P. Elleder M. Patterson M.C. Brady R.O. Vanier M.T. Pentchev P.G. Tagle P.A. Niemann–Pick C1 disease gene: homology to mediators of cholesterol homeostasis.Science. 1997; 277: 228-231Google Scholar). The NPC1 protein is involved in the tubulovesicular trafficking of cholesterol from the late endosome/lysosome to the plasma membrane (PM), and/or to the endoplasmic reticulum (ER) (9Zhang M. Dwyer N.K. Love D.C. Cooney A. Comly M. Neufeld E. Pentchev P.G. Blanchette-Mackie E.J. Hanover J.A. Cessation of rapid late endosomal tubulovesicular trafficking in Niemann-Pick type C1 disease.Proc. Natl. Acad. Sci. USA. 2001; 98: 4466-4471Google Scholar, 10Ko D.C. Gordon M.D. Jin J.Y. Scott M.P. Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events.Mol. Biol. Cell. 2001; 12: 601-614Google Scholar, 11Garver W.S. Krishnan K. Gallagos J.R. Michikawa M. Francis G.A. Heidenreich R.A. Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae.J. Lipid Res. 2002; 43: 579-589Google Scholar, 12Lusa S. Blom T.S. Eskelinen E. Kuismanen E. Mansson J. Simons K. Ikonen E. Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane.J. Cell Sci. 2001; 114: 1893-1900Google Scholar, 13Liscum L. Munn N.J. Intracellular cholesterol transport.Biochim. Biophys. Acta. 1999; 1438: 19-37Google Scholar, 14Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A.M. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. Incardona J.P. Strauss III, J.F. Vanier M.T. Patterson M.C. Brady R.O. Pentchev P.G. Blanchette-Mackie E.J. The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo.J. Biol. Chem. 1999; 274: 9627-9635Google Scholar). These trafficking events may involve the trans-Golgi network/Golgi, and appear to be mediated by Rab proteins (9Zhang M. Dwyer N.K. Love D.C. Cooney A. Comly M. Neufeld E. Pentchev P.G. Blanchette-Mackie E.J. Hanover J.A. Cessation of rapid late endosomal tubulovesicular trafficking in Niemann-Pick type C1 disease.Proc. Natl. Acad. Sci. USA. 2001; 98: 4466-4471Google Scholar, 11Garver W.S. Krishnan K. Gallagos J.R. Michikawa M. Francis G.A. Heidenreich R.A. Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae.J. Lipid Res. 2002; 43: 579-589Google Scholar, 15Higgins M.E. Davies J.P. Chen F.W. Ioannou Y.A. Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network.Mol. Genet. Metab. 1999; 68: 1-13Google Scholar, 16Holtta-Vuori M.J. Maatta J. Ullrich O. Kuismanen E. Ikonen E. Mobilization of late-endosomal cholesterol is inhibited by Rab guanine nucleotide dissociation inhibitor.Curr. Biol. 2000; 10: 95-98Google Scholar, 17Choudhury A. Dominquez M. Puri V. Sharma D.K. Narita K. Wheatley C.L. Marks D.L. Pagano R.E. Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells.J. Clin. Invest. 2002; 109: 1541-1550Scopus (363) Google Scholar). Various glycolipids, including gangliosides, also accumulate in NPC1 cells; the role of NPC1 in causing the accumulation of glycolipids is under active investigation (18Zervas M. Somers K.L. Thrall M.A. Walkley S.U. Critical role for glycosphingolipids in Niemann-Pick disease type C.Curr. Biol. 2001; 11: 1283-1287Google Scholar, 19Taniguchi M. Shinoda Y. Ninomiya H. Vanier M.T. Ohno K. Sites of temporal changes of gangliosides GM1/GM2 storage in the Niemann-Pick disease type C mouse brain.Brain Dev. 2001; 23: 414-421Google Scholar, 20Puri V. Watanabe R. Dominguez M. Sun X. Wheatley C.L. Marks D.L. Pagano R.E. Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases.Nat. Cell Biol. 1999; 1: 386-388Google Scholar). In addition to NPC1 and NPC2, the late endosomal protein MLN 64, an integral membrane protein that contains the cholesterol binding domain StAR, has also been shown to be intimately involved in cholesterol trafficking (21Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss J.F. MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria.J. Biol. Chem. 2002; 277: 33300-33310Google Scholar). Mammalian cells acquire cholesterol via exogenous uptake, mainly from LDL via the LDL receptor pathway. The involvement of NPC1 in distributing LDL-derived cholesterol to the PM and/or to the ER for esterification [by the enzyme acyl-CoA:cholesterol acyltransferase (ACAT)] has been well-recognized [as reviewed in refs. (1Pentchev P.G. Comly M.E. Kruth H.S. Vanier M.T. Wenger D.A. Patel S. Brady R.O. A defect in cholesterol esterification in Niemann–Pick disease (type C) patients.Proc. Natl. Acad. Sci. USA. 1985; 82: 8247-8251Google Scholar, 2Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E.D. Neufeld E.B. Blanchette-Mackie E.J. Pentchev P.G. Niemann–Pick disease type C: a lipid trafficking disorder.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th edition. Vol. 3. McGraw-Hill, New York2001: 3611-3633Google Scholar, 13Liscum L. Munn N.J. Intracellular cholesterol transport.Biochim. Biophys. Acta. 1999; 1438: 19-37Google Scholar)]. In addition to exogenous uptake, cells also acquire cholesterol through endogenous biosynthesis de novo. The involvement of NPC1 in trafficking of endogenously synthesized cholesterol (endoCHOL) has received little attention. Because the major source of cholesterol in the CNS is provided through de novo endogenous cholesterol synthesis (22Turley S.D. Burns D.K. Dietschy J.M. Preferential utilization of newly synthesized cholesterol for brain growth in neonatal lambs.Am. J. Physiol. 1998; 274: E1099-E1105Google Scholar, 23Lutjohann D. Breuer O. Ahlborg G. Nennesmo I. Siden A. Diczfalusy U. Bjorkhem I. Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation.Proc. Natl. Acad. Sci. USA. 1996; 93: 9799-9804Google Scholar), understanding the involvement of NPC1 in the trafficking of endoCHOL remains paramount to elucidating the etiology of NPC disease in the CNS. In cells, cholesterol is synthesized endogenously in the ER, and the majority of this newly synthesized cholesterol quickly moves to the PM within a few minutes, arriving at the cholesterol-rich domain caveolae (24Degrella R.F. Simoni R.D. Intracellular transport of cholesterol to the plasma membrane.J. Biol. Chem. 1982; 257: 14256-14262Google Scholar, 25Urbani L. Simoni R.D. Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane.J. Biol. Chem. 1990; 265: 1919-1923Google Scholar, 26Lange Y. Cholesterol movement from plasma membrane to rough endoplasmic reticulum. Inhibition by progesterone.J. Biol. Chem. 1994; 269: 3411-3414Google Scholar, 27Smart E.J. Ying Y.S. Donzell W.C. Anderson R.G.W. A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane.J. Biol. Chem. 1996; 271: 29427-29435Google Scholar, 28Uittenbogaard A. Ying Y. Smart E.J. Characterization of a cytosolic heat-shock protein-caveolin chaperone complex. Involvement in cholesterol trafficking.J. Biol. Chem. 1998; 273: 6525-6532Google Scholar, 29Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface.Proc. Natl. Acad. Sci. USA. 2000; 97: 8375-8380Google Scholar). This initial movement of newly synthesized cholesterol to the PM is independent of NPC1 (30Liscum L. Ruggiero R.M. Faust J.R. The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts.J. Cell Biol. 1989; 108: 1625-1636Google Scholar, 31Cruz J.C. Chang T.Y. Fate of endogenously synthesized cholesterol in Niemann-Pick type C1 cells.J. Biol. Chem. 2000; 275: 41309-41316Google Scholar). To pursue the post-PM fate of endoCHOL, this laboratory used a Chinese hamster ovary (CHO) cell mutant defective in the NPC1 protein (CT43) and its parental cell line (25RA) as the model system (31Cruz J.C. Chang T.Y. Fate of endogenously synthesized cholesterol in Niemann-Pick type C1 cells.J. Biol. Chem. 2000; 275: 41309-41316Google Scholar, 32Limanek J.S. Chin J. Chang T.Y. Mammalian cell mutant requiring cholesterol and unsaturated fatty acid for growth.Proc. Natl. Acad. Sci. USA. 1978; 75: 5452-5456Google Scholar, 33Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein.Cell. 1996; 87: 415-426Google Scholar). To label the newly synthesized cholesterol, we incubated cells with [3H]acetate, a precursor for cholesterol biosynthesis. To monitor the fate of labeled cholesterol, we performed chase experiments. The results confirmed that the movement of newly synthesized cholesterol from the ER to the PM is independent of NPC1. After initially reaching the PM, significant equilibration of endoCHOL between the PM and the internal membranes takes place within 8 h. In 25RA cells, endoCHOL internalizes from the PM to internal membranes, than recycles back to the PM; endoCHOL also moves to the ER to be esterified by ACAT. When cyclodextrin (CD), a water-soluble molecule that binds to cholesterol with high affinity, was added to the growth medium, the majority of [3H]cholesterol resided in the PM and was largely susceptible to extraction by CD. In contrast, in CT43 cells, a significant portion of the [3H]cholesterol accumulated in the late endosome/lysosome became less available for esterification in the ER and to extraction by CD (31Cruz J.C. Chang T.Y. Fate of endogenously synthesized cholesterol in Niemann-Pick type C1 cells.J. Biol. Chem. 2000; 275: 41309-41316Google Scholar). These studies, along with findings from other investigators (34Lange Y. Ye J. Rigney M. Steck T. Cholesterol movement in Niemann-Pick type C cells and in cells treated with amphiphiles.J. Biol. Chem. 2000; 275: 17468-17475Google Scholar), suggest that irrespective of the origin of cholesterol, the lack of a functional NPC1 protein invariably leads to intracellular cholesterol accumulation, mainly in the late endosome/lysosome. The pathways by which LDL-derived or newly synthesized cholesterol enters the late endocytic pathway may or may not be the same; the cellular nature of these pathways is under investigation in various laboratories. Both the 25RA cells and the CT43 cells contain the same gain-of-function mutation in the protein called SCAP, the SREBP cleavage-activating protein that is involved in the transcription control of many sterol-sensitive genes (33Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein.Cell. 1996; 87: 415-426Google Scholar, 35Goldstein J.L. Rawson R.B. Brown M.S. Mutant mammalian cells as tools to delineate the sterol regulatory element-binding protein pathway for feedback regulation of lipid synthesis.Arch. Biochem. Biophys. 2002; 397: 139-148Google Scholar). It is thus possible that the findings made in 25RA and CT43 cells may not be applicable to other cell types. To test the generality of our findings, in the current work, we examined the role of NPC1 in the intracellular trafficking of endoCHOL in four different cell types isolated from the wild-type, the heterozygous, and the homozygous BALB/c NPC1NIH mice. The cell types include mouse embryonic fibroblasts (MEFs), mouse peritoneal macrophages (MPMs), primary hepatocytes, and cerebellar glial cells. The murine model for the NPC disease, the BALB/c NPC1NIH mouse, has a well-defined mutation in the NPC1 gene, and exhibits a phenotype very similar to that of the human NPC disease (5Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene.Science. 1997; 277: 232-235Google Scholar, 36Dietschy J.M. Turley S.D. Control of cholesterol turnover in the mouse.J. Biol. Chem. 2001; 277: 3801-3804Google Scholar, 37Xie C. Turley S.D. Pentchev P.G. Dietschy J.M. Cholesterol balance and metabolism in mice with loss of function Niemann-Pick C protein.Am. J. Physiol. 1999; 276: E336-E344Google Scholar, 38Bhuvaneswaran C. Morris M.D. Shio H. Fowler S. Lysosomal lipid storage disorder in NCTR/BALB/c mice. III. Isolation and analysis of storage inclusions from liver.Am. J. Pathol. 1982; 108: 160-170Google Scholar, 39Morris M.D. Bhuvaneswaran C. Shio H. Fowler S. Lysosomal lipid storage disorder in NCTR/BALB/c mice. I. Description of the disease and genetics.Am. J. Pathol. 1982; 108: 140-149Google Scholar, 40Voikar V. Rauvala K. Ikonen E. Cognitive deficit and development of motor impairment in a mouse model of Niemann-Pick type C disease.Behav. Brain Res. 2002; 132: 1-10Google Scholar). [3H]acetate (20 Ci/mmol) and [3H]cholesterol (60–90 Ci/mmol) were from American Radiolabeled Chemicals. 2-Hydroxypropyl-β-cyclodextrin, egg phosphatidylcholine, oleic acid, FBS, and trypsin were from Sigma. Medium RPMI-1640, stock solutions of penicillin/streptomycin, trypsin-EDTA, and HBSS were from GibcoBRL. DMEM plus sodium pyruvate and high glucose were from Cellgro. Collagen type I-coated 6-well plates were from Becton Dickinson’s Biocoat. Tissue culture flasks or dishes were from Costar or Falcon. Nylon mesh (35 μm) for hepatocyte isolation was from Small Parts, Inc. (Miami Lakes, FL). Organic solvents were from Fisher. The NPC1 mutant mice (BALB/c NPC1NIH mice) were discovered at the National Center for Toxicological Research, Little Rock, AR (41Morris M.D. Bhuvaneswatan C. Shio H. Fowler S. Lysosome lipid storage disorder in NCTR-BALB/c mice. I. Description of the disease and genetics.Am. J. Pathol. 1982; 108: 140-149Google Scholar), and were generously provided by Peter Pentchev at the National Institutes of Health. NPC1NIH homozygotes are reproductively incompetent because of their neurological impairment and short life span. Heterozygous BALB/c NPC1NIH mice were used to generate the homozygous NPC1 mice and the wild-type (control) mice used in these studies. The genotypes of mice were determined from genomic DNAs isolated from tail snips, using a previously described PCR method (5Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene.Science. 1997; 277: 232-235Google Scholar, 42Henderson L.P. Lin L. Prasad A. Paul C.A. Chang T.Y. Maue R.A. Embryonic striatal neurons from Niemann-Pick type C mice exhibit defects in cholesterol metabolism and neurotrophin responsiveness.J. Biol. Chem. 2000; 275: 20179-20187Google Scholar). All experimental protocols were approved by the Institutional Animal Care and Research Advisory Committee at Dartmouth Medical School and conducted in accordance with the US Public Health Service Guide for the Care and Use of Laboratory Animals. Isolation of MEFs was performed according to a previously described procedure, with minor modifications (43Willnow T.E. Herz J. Genetic deficiency in low density lipoprotein receptor-related protein confers cellular resistance to Pseudomonas exotoxin A. Evidence that this protein is required for uptake and degradation of multiple ligands.J. Cell Sci. 1994; 107: 719-726Google Scholar). Briefly, at E17, mouse embryos were taken out of the uterus of pregnant females from genotype-confirmed heterozygous breeding pairs by cesarean section. For each embryo, the tail and a portion of a limb were removed and prepared for DNA extraction and genotyping. After dissection and incubation in 0.05% trypsin solution, the softened tissue was disrupted by repeated pipetting. Cell debris was separated, and the supernatants containing dissociated cells were plated in 25 cm2 flasks, and grown for five successive passages before cells were used for experiments. Isolation of mouse resident (nonstimulated) peritoneal macrophages was performed according to the procedure previously described (44Kim J.G. Keshava C. Murphy A.A. Pitas R.E. Parthasarathy S. Fresh mouse peritoneal macrophages have low scavenger receptor activity.J. Lipid Res. 1997; 11: 2207-2215Google Scholar), from 7- to 8-week-old genotype-confirmed mice via peritoneal lavage. Isolation of mouse primary hepatocytes was performed via a two-stage liver perfusion protocol according to the procedure previously described (45Klaunig J.E. Goldblatt P.J. Hinton D.E. Lipsky M.M. Chacko J. Trump B.F. Mouse liver cell culture. I. Hepatocyte isolation.In Vitro. 1981; 10: 913-925Google Scholar, 46Klaunig J.E. Goldblatt P.J. Hinton D.E. Lipsky M.M. Trump B.F. Mouse liver cell culture. II. Primary culture.In Vitro. 1981; 10: 926-934Google Scholar), from 7- to 8-week-old genotype-confirmed mice. Isolation of glial populations was performed according to the procedure previously described (47Noble M. Mayer-Proschel M. Culture of astrocytes, oligodendrocytes, and O-2A progenitor cells.in: Banker G. Goslin K. Culturing Nerve Cells. 2nd edition. MIT Press, Cambridge, MA1998: 499-544Google Scholar), with minor modifications. Briefly, cerebellums from 3- to 5-day-old genotype-confirmed animals were isolated and digested with collagenase (at 37°C for 30 min) and then with trypsin (at 37°C for 2 min) as described; the resultant cell homogenates were plated into 75 cm2 flasks. To purify the cell population, the glial populations were grown for five successive passages before they were used for experiments. At this point the glial populations were comprised of at least 95% astrocytes, with microglia, oligodendroglia, and their precursor cells comprising less than 5% of the total cell population (47Noble M. Mayer-Proschel M. Culture of astrocytes, oligodendrocytes, and O-2A progenitor cells.in: Banker G. Goslin K. Culturing Nerve Cells. 2nd edition. MIT Press, Cambridge, MA1998: 499-544Google Scholar). MEFs (between passages 5–15) were maintained in DMEM containing 10% FBS in 75 cm2 flasks. For experiments, they were seeded into 6-well plates. Freshly isolated macrophages were seeded at 1 × 106 cells into 6-well plates or 25 cm2 flasks, in RPMI-1640 containing 10% FBS for experiments. After plating, they were allowed to adhere for 2 h; the nonadherent cells were washed away, and fresh media were added. Freshly isolated hepatocytes were seeded at 1 × 106 cells into collagen type I-coated 6-well plates in DMEM containing 10% FBS supplemented with 0.1 μM insulin and 0.1 μM dexamethasone. Hepatocytes were allowed to adhere for 4 to 6 h; nonadherent cells were washed away, and fresh media were added. Cell viability, tested via trypan-blue exclusion, was greater than 95%. Glial cells were maintained in 75 cm2 flasks in DMEM containing 10% FBS, seeded into 6-well plates or 25 cm2 flasks for experiments. All cell types were maintained in a humidified incubator at 37°C with 10% CO2. Media with 10% FBS is referred to as Media A. The delipidated media used in various experiments, referred to as Medium D, consists of the same base medium used for each cell type, with the 10% FBS replaced with 5% delipidated FBS and 35 μM oleic acid. The delipidated FBS was prepared as described previously (48Chin J. Chang T.Y. Evidence for coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and low density lipoprotein binding activity.J. Biol. Chem. 1981; 256: 6304-6310Google Scholar). All media contained penicillin/streptomycin. On day 1, cells were seeded into 6-well plates and cultured in 2 ml Medium A/well for 24 h. On day 2, cells were washed once with prewarmed PBS (for MEF/MPM) or HBSS (for hepatocytes), and then fed with 2 ml/well of Medium D/well for 48 h. On day 4, cells were washed once with PBS/HBSS, and then subjected to various cholesterol trafficking assays as follows. This assay examines the efflux of endoCHOL to CD within 1 h after the newly synthesized cholesterol arrives at the PM. The cells were labeled with 20 μCi [3H]acetate/well for 1 h at 37°C in Medium D, washed four times, and incubated in Medium D containing 4% CD for 0–30 min. At each time point, the media, along with two PBS washes, were collected. The cells were harvested in 0.2 N NaOH; HCl and phosphate buffer were then added to neutralize the cell lysates. The media and cell lysates were separately extracted with chloroform-methanol (2:1; v/v); the radiolabeled lipids were separated via thin-layer chromatography (TLC) and measured in a liquid scintillation counter as described previously (31Cruz J.C. Chang T.Y. Fate of endogenously synthesized cholesterol in Niemann-Pick type C1 cells.J. Biol. Chem. 2000; 275: 41309-41316Google Scholar). This assay examines the efflux of endoCHOL to CD after significant equilibration of endoCHOL between the PM and the internal membranes takes place. The cells were labeled with 20 μCi [3H]acetate/well for 8 h at 37°C and chased in Medium D for 0 h or 16 h at 37°C. They were washed and then treated with Medium D containing 4% CD for 10 min at 37°C before being harvested and analyzed as described above. Here we utilized a short CD incubation period because we have found that short incubation periods with CD (≤10 min) remove cholesterol predominantly from the PM without inducing the efflux of cholesterol from internal membranes (results to be published from this laboratory). This assay examines the esterification of endoCHOL after a longer labeling period (8 h). The cells were labeled with [3H]acetate in the same manner as described in Assay B. After labeling, cells were washed four times with buffer, and were either harvested in NaOH immediately (for the 0 h chase time point), or chased in Medium D for 16 h then harvested in NaOH (for the 16 h-chase time point). Labeled cholesteryl esters were extracted and analyzed as described previously (31Cruz J.C. Chang T.Y. Fate of endogenously synthesized cholesterol in Niemann-Pick type C1 cells.J. Biol. Chem. 2000; 275: 41309-41316Google Scholar). The percent of cholesterol efflux and percent of cholesterol esterification were calculated as previously described (31Cruz J.C. Chang T.Y. Fate of endogenously synthesized cholesterol in Niemann-Pick type C1 cells.J. Biol. Chem. 2000; 275: 41309-41316Google Scholar). Previous results showed that when 35 μM oleic acid was included in the medium, more than 98% of the 3H present in the labeled cholesteryl ester derived from [3H]acetate resided in the cholesterol moiety rather than the fatty acid moiety. This assay examines the esterification of cell surface-labeled cholesterol. The cells were labeled with [3H]cholesterol containing liposome (49Tabas I. Rosoff W.J. Boykow G.C. A" @default.
• W2100904923 created "2016-06-24" @default.
• W2100904923 creator A5013353605 @default.
• W2100904923 creator A5047179498 @default.
• W2100904923 creator A5072490126 @default.
• W2100904923 date "2003-05-01" @default.
• W2100904923 modified "2023-10-15" @default.
• W2100904923 title "Trafficking defects in endogenously synthesized cholesterol in fibroblasts, macrophages, hepatocytes, and glial cells from Niemann-Pick type C1 mice" @default.
• W2100904923 cites W1507071836 @default.
• W2100904923 cites W1508282658 @default.
• W2100904923 cites W1517698792 @default.
• W2100904923 cites W1518063279 @default.
• W2100904923 cites W1521683984 @default.
• W2100904923 cites W1527093201 @default.
• W2100904923 cites W1537548031 @default.
• W2100904923 cites W1581881472 @default.
• W2100904923 cites W1690219722 @default.
• W2100904923 cites W1940329105 @default.
• W2100904923 cites W1961174746 @default.
• W2100904923 cites W1965589422 @default.
• W2100904923 cites W1967516512 @default.
• W2100904923 cites W1976252335 @default.
• W2100904923 cites W1983606596 @default.
• W2100904923 cites W1985294414 @default.
• W2100904923 cites W1987726242 @default.
• W2100904923 cites W1993629125 @default.
• W2100904923 cites W1994312681 @default.
• W2100904923 cites W1995292245 @default.
• W2100904923 cites W2007406550 @default.
• W2100904923 cites W2012008185 @default.
• W2100904923 cites W2013199292 @default.
• W2100904923 cites W2016152013 @default.
• W2100904923 cites W2026379387 @default.
• W2100904923 cites W2036078107 @default.
• W2100904923 cites W2037373871 @default.
• W2100904923 cites W2039202215 @default.
• W2100904923 cites W2042102113 @default.
• W2100904923 cites W2042663108 @default.
• W2100904923 cites W2049312906 @default.
• W2100904923 cites W2051988120 @default.
• W2100904923 cites W2053203314 @default.
• W2100904923 cites W2057365200 @default.
• W2100904923 cites W2057938901 @default.
• W2100904923 cites W2058576283 @default.
• W2100904923 cites W2066812102 @default.
• W2100904923 cites W2072024204 @default.
• W2100904923 cites W2076246718 @default.
• W2100904923 cites W2081340046 @default.
• W2100904923 cites W2081458493 @default.
• W2100904923 cites W2088432812 @default.
• W2100904923 cites W2090972365 @default.
• W2100904923 cites W2091497761 @default.
• W2100904923 cites W2096221379 @default.
• W2100904923 cites W2096330132 @default.
• W2100904923 cites W2108660588 @default.
• W2100904923 cites W2126500773 @default.
• W2100904923 cites W2134893181 @default.
• W2100904923 cites W2136977521 @default.
• W2100904923 cites W2137387260 @default.
• W2100904923 cites W2141349774 @default.
• W2100904923 cites W2168016965 @default.
• W2100904923 cites W2169810408 @default.
• W2100904923 cites W2307447599 @default.
• W2100904923 cites W2346880178 @default.
• W2100904923 cites W2402457099 @default.
• W2100904923 cites W2419444252 @default.
• W2100904923 cites W4235887954 @default.
• W2100904923 cites W4238609105 @default.
• W2100904923 doi "https://doi.org/10.1194/jlr.m300009-jlr200" @default.
• W2100904923 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12611909" @default.
• W2100904923 hasPublicationYear "2003" @default.
• W2100904923 type Work @default.
• W2100904923 sameAs 2100904923 @default.
• W2100904923 citedByCount "52" @default.
• W2100904923 countsByYear W21009049232013 @default.
• W2100904923 countsByYear W21009049232014 @default.
• W2100904923 countsByYear W21009049232015 @default.
• W2100904923 countsByYear W21009049232016 @default.
• W2100904923 countsByYear W21009049232017 @default.
• W2100904923 countsByYear W21009049232018 @default.
• W2100904923 countsByYear W21009049232019 @default.
• W2100904923 countsByYear W21009049232020 @default.
• W2100904923 countsByYear W21009049232021 @default.
• W2100904923 countsByYear W21009049232023 @default.
• W2100904923 crossrefType "journal-article" @default.
• W2100904923 hasAuthorship W2100904923A5013353605 @default.
• W2100904923 hasAuthorship W2100904923A5047179498 @default.
• W2100904923 hasAuthorship W2100904923A5072490126 @default.
• W2100904923 hasBestOaLocation W21009049231 @default.
• W2100904923 hasConcept C134018914 @default.
• W2100904923 hasConcept C185592680 @default.
• W2100904923 hasConcept C202751555 @default.
• W2100904923 hasConcept C2778163477 @default.
• W2100904923 hasConcept C2779489717 @default.
• W2100904923 hasConcept C2780381497 @default.
• W2100904923 hasConcept C2780884517 @default.
• W2100904923 hasConcept C529278444 @default.
• W2100904923 hasConcept C55493867 @default.

• next